Mixed anhydrides of N&#39;-acetylaminothioalkanoates

ABSTRACT

N-acetyl-para-aminophenyl N&#39;-acetylaminothioalkanoates I are new analgesic compounds with greatly reduced hepatotoxic effects, when taken in overdose, relative to N-acetyl-para-aminophenol. They are prepared by reacting an N-acetylaminothioalkanoic acid IV with a reactive organic chloride V to form a mixed anhydride II and then reacting the latter with N-acetyl-para-aminophenol. The mixed anhydrides II are new and useful intermediates. Alternatively the derivatives I may be prepared by reacting the acid IV with bis-(4-nitrophenyl) sulfite to form a para-nitrophenyl N-acetylaminothioalkanoic acid ester VIII, reducing the latter to a para-aminophenyl N-acetylaminothioalkanoate VII, and acetylating this product. The esters VII and VIII are new and useful intermediates. Both reactions may pass through S-blocked intermediates, which are also new. Pharmaceutical compositions containing the derivatives I are disclosed, and also analgesic methods using them.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of application Ser. No. 909,846, filedMay 26, 1978, now U.S. Pat. No. 4,181,719, issued Jan. 1, 1980.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is concerned with analgesic compounds, processes formaking them and the compounds thus prepared, various intermediatesuseful in their synthesis, and pharmaceutical compositions incorporatingthese analgesic compounds. More especially, the invention relates toN-acetyl-para-aminophenyl N'-acetylaminothioalkanoates which are usefulas analgesic agents having analgesic activity like that ofN-acetyl-para-aminophenol but being largely free from the hepatotoxiceffects normally encountered when N-acetyl-para-aminophenol is taken inoverdose.

2. Description of the Prior Art

The compound N-acetyl-para-aminophenol, also known as acetaminophen,paracetamol, para-hydroxyacetanilide, APAP, NAPA, etc., is a widely usedanalgesic agent. Unfortunately the toxic nature of the drug when takenin overdose has been given wide publicity; and coupled with its readyavailability this has led to the increasing use of this drug in suicide.The major organ affected by an overdose of N-acetyl-para-aminophenol isthe liver; and even when an overdose is not fatal, it can cause severeliver damage.

Although this invention is not limited by any theoreticalconsiderations, the following background explanation is offered. It isthought that hepatic damage induced by N-acetyl-para-aminophenol isrelated to the metabolism of the drug converting it into a highlychemically-reactive derivative, which binds covalently to liver cellproteins when the drug is given in overdose; whereas non-hepatotoxicdoses of N-acetyl-para-aminophenol give rise to little or no convalentbinding. Although the exact nature of the toxic metabolite is not known,it is thought to be a precursor of the cysteine and mercapturic acidconjugates of N-acetyl-para-aminophenol, as excretion of theseconjugates increases significantly in the overdose situation.

The cysteine and mercapturic acid conjugates ofN-acetyl-para-aminophenol are formed via the activation of the parentmolecule to a chemically-unstable intermediate, which normally willimmediately form a complex with reduced glutathione within the livercell. In the overdose situation, however, activation ofN-acetyl-para-aminophenol occurs faster than hepatic reduced glutathionecan be synthesized; and as the level of glutathione falls, some of thereactive metabolite combines with hepatocyte proteins instead. Thequantity of the chemically-unstable intermediate convalently bound tohepatocyte proteins correlates with the severity of the ensuing liverdamage.

In the overdose situation, the logical antidote would therefore seem tobe glutathione itself; but, if administered exogenously, glutathionefails to penetrate the cells, and is therefore ineffective unlessmassive doses are administered. Instead therefore it is conventional totreat overdoses with glutathione substitutes--sulfhydrylcompounds--e.g., cysteamine or dimercaprol. It has been suggested thatcysteamine may inhibit oxidation of N-acetyl-para-aminophenol to thetoxic metabolite.

There is also a less obvious way of tackling the same problem. While anN-acetyl-para-aminophenol overdose results in glutathione depletion, itshould be remembered that glutathione synthesis is still takingplace--so by providing a glutathione precursor it should be possible topromote glutathione synthesis, thus to reduce the rate at which theglutathione levels fall, and in this way to keep the glutathione levelsfrom falling to the low level at which the activeN-acetyl-para-aminophenol metabolite starts to attack the hepatocyteproteins. Here again therefore it is conventional to treat overdoses ofN-acetyl-para-aminophenol by providing a glutathione precursor, e.g.methionine or cysteine.

These conventional methods of treating overdoses ofN-acetyl-para-aminophenol are however often ineffective, due to the timelapse between ingestion of the N-acetyl-para-aminophenol overdose andthe arrival of the glutathione substitute or glutathione precursor inthe liver cells.

The following references are summarized to show the state of the priorart:

A. E. M. McLean, THE LANCET, p. 729 (Apr. 20, 1974), first showed thecombination of methionine with paracetamol as a way of greatly reducingthe lethal and hepatotoxic effects of paracetamol overdosage. McLeanconcludes: "Methionine seems an easy way in which we can makeparacetamol a safer drug" and also suggests the use of cystine as asubstitute for methionine.

Maxwell et al., THE LANCET, pp. 610-611 (Sept. 27, 1975), reported thatthe above-noted McLean's study "demonstrated the decreased toxicity ofthe combination of acetaminophen and methionine in rats" and that "ourstudy demonstrates it in dogs".

McLean and Day, "The Effect of Diet on the Toxicity of Paracetamol andthe Safety of Paracetamol-Methionine Mixtures", Biochemical Pharmacology24, 37-42 (1975)--The abstract reads in part: "Addition of methionine tooral paracetamol dose protects against death and liver injury, and it issuggested that this may be a useful technique for making paracetamolsafe against the danger of overdose."

The National Research Development Corp. German patent application No.2,455,203, published May 22, 1975, (based on application No. 455,203,filed Nov. 21, 1974, in turn, based on British application No. 54,098,filed Nov. 21, 1973) is abstracted in the July 8, 1975 DERWTNT® GERMANPATENTS GAZETTE as follows:

"Medicaments contg. p-hydroxyacetanilide (I) as active ingredient alsocontain a cpd. (II) which is a precursor of the central amino acidgrouping of glutathione and which can be converted into glutathione invivo. (I) is widely used as an analgesic and antipyretic, but overdosesgive rise to liver necrosis associated with the absence of glutathionein the liver; if an overdose of (I)+(II) is taken, this automaticallyleads to an increase in the glutathione synthesised in the organism,thus counteracting the toxic effects of the overdose. The glutathioneprecursor (II) can be an S-contg. amino acid, pref. DL- or L-cysteine,-cystine or -methionine (esp. the L-form), or a di- or tripeptide contg.such an amino acid. The medicament pref. contains 10-50% (esp. 10-30%)of (II), based on the wt. of (I). The medicaments are pref. administeredorally (esp. as elixirs or tablets) in a unit dose of 0.25-1 g."

The Italchemi S.r.l.-Instituto Chimico Farmaceutico French BSM No.4672M, published Jan. 23, 1967, shows as anti-inflammatory,anti-allergic, analgesic and antipyretic agents the paracetamol estersof gamma-aminobutyric acid, delta-aminovaleric acid andepsilon-aminocaproic acid and their N-acetyl derivatives.

Kovach, "Aminoacid esters of p-acetamidophenol as prodrugs", a Ph.D.thesis, University of Kansas, 1974 [C.A. 83, 193681t (1975); Diss.Abstr. Int. B 1975, 36(2), 734-5]--The abstract of this thesis shows thepreparation of paracetamol (APAP) esters of amino acids, specificallyglycine, alpha-aspartic acid and beta-aspartic acid.

Piperno et al. (McNeil Labs. Inc., Fort Washington, Pa.), THE LANCET,pp. 738-739 (Oct. 2, 1976)--Letter to the editor with the title,"Reversal of Experimental Paracetamol Toxicosis with N-Acetylcysteine".The authors conclude that their data "suggest that N.A.C. sodium(N-acetylcysteine sodium) will be effective orally and parenterally inthe clinic at doses approximately to 20-40% of the ingested amount ofparacetamol".

Gerber et al. (Dept. of Pharmacology, Vanderbilt University, School ofMedicine, Nashville, Tenn.), THE LANCET, pp. 657-658 (Mar. 19,1977)--Letter to the editor with heading: "Effect of N-Acetylcysteine onHepatic Convalent Biding or Paracetamol (Acetaminophen)". Gerber et al.reported that" 1 g./kg. of N-acetylcysteine administered concurrently or2 or 4 hours after 1.4 g./kg. of paracetamol "significantly improved 48hour survival in mice".

Mead Johnson & Co. Sheffner U.S. Pat. No. 3,591,686, issued July 6, 1971(based on application Ser. No. 657,498, filed Aug. 1, 1967, in turn acontinuation in part of application Ser. No. 482,931, filed Aug. 26,1965) has "method-of-treating" and composition claims covering the useof N-acetylcysteine and related "--(N-acylamino)-β-mercaptoalkanoicacids or salts thereof" as anti-inflammatory agents.

SUMMARY OF THE INVENTION

The present invention is based partly upon the premise that bychemically combining a glutathione precursor withN-acetyl-para-aminophenol it ought to be possible to minimize the dangerof liver damage in the overdose situation--for the act of ingesting anoverdose of N-acetyl-para-aminophenol should then ipso facto ensure thatthe sulfhydryl groups needed in a glutathione precursor willsimultaneously be present in the liver to help replenish the glutathionestores.

Important though it is, that is not the only advantage of the presentinvention. We have also found that the chemical combination ofN-acetyl-para-aminophenol with a glutathione precursor possesses anextremely palatable flavor, far more acceptable than the extremelybitter taste of the former, and is moreover free from the pronouncedodor of the latter.

According to one aspect of this invention there are therefore provided,as new compounds of analgesic activity but relatively free from relatedhepatotoxic effects when taken in overdose, theN-acetyl-para-aminophenyl N'-acetylaminothioalkanoates which conform tothe general formula I ##STR1## in which n is 1 or 2; and X is a hydrogenatom, a methyl group or a grouping of general formula I' ##STR2##wherein n' is 1 or 2; and p is zero or 1.

Specific preferred N-acetyl-para-aminophenylN'-acetylaminothioalkanoates of general formula I above, for use inaccordance with this invention, are for instance as follows:

N-Acetyl-para-aminophenyl N'-acetyl-methionate of the structuralformula: ##STR3## (conforming to general formula I above, where n is 2and X is a methyl group);

N-Acetyl-para-aminophenyl N'-acetyl-cysteinate of the structuralformula: ##STR4## (conforming to general formula I above, where n is 1and X is a hydrogen atom);

Di-(N-acetyl-para-aminophenyl) N',N"-diacetyl-cystinate of structuralformula: ##STR5## (conforming to general formula I above, where n is 1and X is the grouping of general formula I' where p is 1 and n' is 1);

Di-(N-acetyl-para-aminophenyl) N',N"-diacetylcystathionate of structuralformula: ##STR6## (conforming to general formula I above, where n is 1and X is the grouping of general formula I' wherein p is 0 and n' is 2);

N-Acetyl-para-aminophenyl N'-acetyl-S-methylcysteinate of the structuralformula: ##STR7## (conforming to general formula I above, where n is 1and X is a methyl group); and

N-Acetyl-para-aminophenyl N'-acetyl-homocysteinate of the structuralformula: ##STR8## (conforming to general formula I above, where n is 2and X is a hydrogen atom).

All the N-acetyl-para-aminophenyl N'-acetylaminothioalkanoates ofgeneral formula I above, and specifically the preferred ones ofstructural formula A-F above, include at least one asymmetric carbonatom, and therefore exist as separate optically-active D and L isomers,as well as in the form of mixtures of the D and L isomers, above all asthe equiproportioned racemic mixture in which the compounds willnormally be prepared unless optically-active starting materials areemployed in the processes described below. This invention of courseextends to all the compounds of formula I as individual optically-activeD and L isomers and also in the form of mixtures thereof, especially atracemic mixtures. Because it is the L-amino acids which occur naturallyin the body, we prefer when economic considerations permit to use theL-isomers of the compounds of this invention, thus for instanceN-acetyl-para-aminophenyl N'-acetyl-L-cysteine andN-acetyl-para-aminophenyl N'-acetyl-L-methionate.

We have moreover found that these N-acetyl-para-aminophenylN'-acetylaminothioalkanoates of general formula I above, andspecifically the preferred ones of structural formulae A-F above, can beconveniently prepared, usually in good yield, by various alternativeprocedures.

According to another aspect of this invention there is thereforeprovided a process for preparing the N-acetyl-para-aminophenylN'-acetylaminothioalkanoates of general formula I above, which includesthe step of reacting a mixed anhydride of general formula II ##STR9##wherein n is 1 or 2; m is zero or 1; Q represents either a carbon atom Cor when m is zero a sulfinyl group S═O; "Ayl" represents an alkyl groupwith 1-20 carbon atoms, an aryl group with 6-20 carbon atoms, an alkarylgroup with 7-20 carbon atoms or an aralkyl group with 7-20 carbon atoms;and X' is a hydrogen atom, a blocking group, a methyl group, or a mixedanhydride group conforming to the general formula II' ##STR10## in whichQ and Ayl both have their previously-indicated meanings; n' is 1 or 2';m' is zero or 1; and p is zero or 1, with N-acetyl-para-aminophenol toform the corresponding N-acetyl-para-aminophenylN'-acetylaminothioalkanoate, and thereafter when X' is a blocking groupthis is removed, thus yielding the desired correspondingN-acetyl-para-aminophenyl N'-acetylaminothioalkanoate of general formulaI.

The reaction between the mixed anhydride II andN-acetyl-para-aminophenol can and preferably should be performed in ananhydrous inert organic solvent. For reasons which will appear below itshould be noted that this reaction can moreover be conducted withoutdetriment in the presence of a base, such as a tertiary amine liketriethylamine or pyridine.

The invention extends to the N-acetyl-para-aminophenylN'-acetyl-aminothioalkanoates formed in the course of theabove-described reaction wherein X' is a blocking group, thus thosewhich conform to the general formula III ##STR11## in which n is 1 or 2;and "Block" represents a blocking group or a mixed anhydride groupconforming to the general formula III' in which n' is 1 or 2; and p iszero or 1, which are new intermediates useful in synthesis.

The mixed anhydrides of general formula II used as starting material inthe above-described reaction may advantageously be prepared in apreliminary stage by reacting an N'-acetylaminothioalkanoic acid orblocked derivative thereof of general formula IV ##STR12## in which n is1 or 2; and X' is a hydrogen atom, a blocking group, a methyl group or agrouping of general formula IV' ##STR13## wherein n' is 1 or 2' and p iszero or 1, or a salt thereof with a reactive organic chloride of generalformula ##STR14## in which "Ayl" represents an alkyl group with 1-20carbon atoms, an aryl group with 6-20 carbon atoms, an alkaryl groupwith 7-20 carbon atoms or an aralkyl group with 7-20 carbon atoms; m iszero or 1; and Q represents either a carbon atom C or when m is zero asulfinyl group S═O, in the presence of a base to yield the desiredcorresponding mixed anhydride of general formula II.

The reactive organic chloride of general formula V above can forinstance be an alkyl, aralkyl, aryl or alkaryl chloroformate of generalformula Va ##STR15## or a sterically-encumbered alkyl, aralkyl, aryl oralkaryl acid chloride of general formula Vb ##STR16## or an alkyl,aralkyl, aryl or alkaryl sulfonyl chloride of general formula Vc##STR17## in all of which general formulae Va, Vb and Vc "Ayl"represents an alkyl, aralkyl, aryl or alkaryl group having up to 20carbon atoms therein.

The reaction (sometimes hereinafter called the "first-stage reaction")between the aminothioalkanoic acid (or blocked derivative thereof) ofgeneral formula IV or a salt thereof and the reactive chloride ofgeneral formula V (or Va, Vb or Vc) is most conveniently carried out inan anhydrous inert and usually organic solvent, preferably a polarorganic solvent, for example, either a substituted aromatic solvent suchas toluene or any halogenated aromatic hydrocarbon, or most desirably ahalogenated aliphatic hydrocarbon such as dichloromethane or above allchloroform (trichloromethane).

This first-stage reaction must be performed in the presence of a base,which advantageously will be a tertiary amine, and preferably eithertriethylamine or pyridine.

As previously indicated, the subsequent reaction between the mixedanhydride and N-acetyl-para-aminophenol (sometimes hereinafter calledthe "second-stage reaction") is advantageously performed in the sameanhydrous inert organic solvent as that in which the first-stagereaction is performed, and the second-stage reaction moreover suffers nodetriment from the presence of base. It is therefore possible and indeeddesirable to carry out both the first-stage reaction and thesecond-stage reaction in the same anhydrous inert organic solvent,without intervening isolation of the first-stage reaction product, andusually in the same reaction vessel.

This invention extends to the blocked N'-acetyl-aminothioalkanoic acidderivatives IV which may be used as starting material in the first-stagereaction just described, thus those which conform to the general formulaVI ##STR18## in which n is 1 or 2; and "Block" represents a blockinggroup which are new intermediates useful in synthesis.

In cases where it is desired to prepare an N-acetyl-para-aminophenylN'-acetylaminothioalkanoate of general formula I wherein X is a hydrogenatom, it may often be advisable, and sometimes indeed may be necessary,to employ a blocked N'-acetylaminothioalkanoic acid derivative VI asstarting material in the first-stage reaction, so as to protect the S--Hbond from attack during that and the subsequent second-stage reaction.This can be effected in a manner well known and understood in the art,by substituting the hydrogen of the S--H bond with a blocking group, forinstance, tetrahydro-2-pyranyl, from dihydropyran.

In the preparation of an N-acetyl-para-aminophenylN'-acetylaminothioalkanoate of general formula I wherein X is a hydrogenatom, the overall process should therefore desirably include the initialstep of preparing the blocked N'-acetylaminothioalkanoic acid derivativeVI for use therein by substituting the S--H bond of anN'-acetylaminothioalkanoic acid of general formula IV wherein X ishydrogen with a blocking group before the first-stage reaction with thereactive organic chloride V, and also include the final step ofreplacing the blocking group in the correspondingN-acetyl-para-aminophenyl N'-acetylaminothioalkanoate with a hydrogenatom after the reaction with N-acetyl-para-aminophenol.

The blocking group used is preferably one derived from dihydropyran,that is, tetrahydro-2-pyranyl.

According to a still further aspect of this invention there is alsoprovided an alternative process for preparing theN-acetyl-para-aminophenyl N'-acetylaminothioalkanoates of generalformula I, which includes the step of reacting a para-aminophenylN-acetylaminothioalkanoate of general formula VII ##STR19## in which n"is 1 or 2' and X" is a hydrogen atom, a blocking group, a methyl groupor a grouping of the general formula VII' ##STR20## wherein n"' is 1 or2; and p is zero or 1, with an acetylating agent to yield thecorresponding N-acetyl-para-aminophenyl N'-acetylaminothioalkanoate, andthereafter when X" is a blocking group this is removed, thus yieldingthe desired corresponding N-acetyl-para-aminophenylN'-acetylaminothioalkanoate of general formula I.

The acetylation of the para-aminophenyl N-acetylaminothioalkanoate VIIcan be performed using any convenient acetylating agent, such aspreferably acetic anhydride.

The invention extends to the para-aminophenylN-acetylaminothioalkanoates VII used as starting material in thereaction just described, which are new intermediates useful insynthesis.

These para-aminophenyl N-acetylaminothioalkanoates VII can be preparedin a preliminary stage of the process described immediately above byreacting a para-nitrophenyl N'-acetylaminothioalkanoate of generalformula VIII ##STR21## in which n" is 1 or 2; and X" is a hydrogen atom,a blocking group, a methyl group or an ester grouping conforming to thegeneral formula VIII' ##STR22## wherein n"' is 1 or 2; and p is zero or1, with a reducing agent.

The reduction of the para-nitrophenyl N-acetylaminothioalkanoate VIIIcan best be carried out using nascent hydrogen as the reducing agent,which can most conveniently be by iron powder and acid, preferably hotacetic acid.

The invention extends to the para-nitrophenylN'-acetylaminothioalkanoates VIII used as starting materials in thepreliminary stage of the process just described, which are newintermediates useful in synthesis.

These para-nitrophenyl N'-acetylaminothioalkanoates VIII can in turn beprepared in an initial stage of this alternative process by reacting anN-acetylaminothioalkanoic acid of general formula IV withbis-(4-nitrophenyl) sulfite.

The reaction between the N-acetylaminothioalkanoic acid IV and thebis-(4-nitrophenyl) sulfite is advantageously carried out in solution ina tertiary amine, preferably pyridine; and it can and convenientlyshould be carried out at ambient or near-ambient temperature.

As with the other, first-described process, in cases where it is desiredto prepare an N-acetyl-para-aminophenyl N'-acetylaminothioalkanoate ofgeneral formula I where X is a hydrogen atom, it may often be advisable,and sometimes indeed it may be necessary, to employ a blockedN'-acetylaminothioalkanoic acid, thus one of general formula VI, as thestarting material above, so as to protect the S--H bond from attachduring the subsequent reaction stages. As before, this can be effectedin a manner well known and understood in the art, by substituting thehydrogen of the S--H bond with a blocking group, for instance,tetrahydropyranyl.

In the preparation of an N-acetyl-para-aminophenylN'-acetylaminothioalkanoate of general formula I wherein X is a hydrogenatom, this alternative process overall should therefore desirablyinclude the initial step of preparing the blocked derivative ofN-acetylaminothioalkanoic acid of general formula VI for use therein bysubstituting the S--H bond of an N'-acetylaminothioalkanoic acid esterderivative of general formula IV wherein X is hydrogen with a blockinggroup before reaction with the bis-(4-nitrophenyl) sulfite, and alsoinclude the final step of replacing the blocking group in thecorresponding N-acetyl-para-aminophenyl N'-acetylaminothioalkanoate witha hydrogen atom after the reaction with the acetylating agent.

As already indicated above, the blocking group used will preferably betetrahydro-2-pyranyl, which is derived from dihydropyran. Of course,other blocking groups capable of blocking or protecting thiols can beused provided the blocked thiol, e.g., thioether, thioacetal,thiolester, etc., can be readily reconverted to the thiol.

Preferred groups for "Ayl", as used herein, e.g., in the definition ofthe compounds of formula II, include: methyl, ethyl, n-propyl,isopropyl, n-butyl, 2-butyl and isobutyl when "alkyl"; phenyl,2-chlorophenyl, 3-chlorophenyl and 4-chlorophenyl when "aryl"; benzylwhen "aralkyl"; and, ortho-tolyl, meta-tolyl and para-tolyl when"alkaryl".

For employment in human medicine as an analgesic theN-acetyl-para-aminophenyl N'-acetylaminothioalkanoates of this inventionshould, of course, be suitable formulated into pharmaceuticalcompositions by association with suitable pharmaceutical vehicles.

The term "pharmaceutical" is used herein to exclude any possibility thatthe nature of the vehicle (considered, of course, in relation to theroute by which the composition is intended to be administered) could beharmful, rather than beneficial. The choice of a suitable vehicle forany chosen route and mode of presentation is believed to be within thecompetence of those accustomed to the preparation of pharmaceuticalcompositions.

Accordingly, in yet another aspect of this invention, there are providedpharmaceutical compositions containing one or more of theN-acetyl-para-aminophenyl N'-acetylaminothioalkanoates of generalformula I above in association with a suitable pharmaceutical vehicle asherein defined.

The compositions of this invention may be administered orally orrectally. In respect of these various modes of presentation the"pharmaceutical vehicle" is preferably:

(a) the ingestible excipient of a tablet, coated tablet, or pill; theingestible container of a capsule or cachet; the ingestible pulverulentsolid carrier of a powder; or the ingestible liquid medium of a syrup,solution, suspension or elixir; or

(b) a base material of low melting point capable of releasing the activeingredient to perform its pharmacological function, which base material,when appropriately shaped, forms a suppository.

While the modes of presentation just listed represent those most likelyto be employed, they do not necessarily exhaust the possibilities.

The N-acetyl-para-aminophenyl N'-acetylaminothioalkanoates of thisinvention will most frequently be administered in the form of tablets,capsules and other solid unit-dose forms. Bearing in mind the dosagerates recommended below it will frequently be desirable to employ thecomposition in the form of solid unit-doses containing substantially 500mg. of the N-acetyl-para-aminophenyl N'-acetylaminothioalkanoic acidester derivatives.

While the dosage rates for the analgesic N-acetyl-para-aminophenylN'-acetylaminothioalkanoates of general formula I above will, to acertain degree, depend upon the route by which the compositions are tobe administered, and of course also upon the condition to be treated, itcan as a general indication be said that the useful dose normally willbe within the range of from 1 g. to 4 g. of the active analgesic per dayfor an adult.

It may conveniently here be noted that the N-acetyl-para-aminophenolN'-acetylaminothioalkanoates of general formula I above can of course beadministered in medicine and where appropriate be formulated intopharmaceutical compositions for that purpose, in conjunction with otherpharmacologically-active ingredients, such as for instance one or moreof the following, namely N-acetyl-para-aminophenol, codeine, codeinephosphate, caffeine, caffeine hydrate, caffeine citrate, butobarbitone,famprofazone, propyphenazone, chlormezanone, phenolphthalein, aspirin,acetomenaphthone, orphenadrine citrate, phenobarbitone, dihydrocodeinetartrate, dextropropoxyphene and pseudoephedrine hydrochloride. Ofcourse, it will be realized that the relative freedom from hepatotoxiceffects enjoyed by the N-acetyl-para-aminophenylN'-acetylaminothioalkanoates of general formula I above will not extendto any such overdoses of the other ingredients.

In order that the invention may be well understood, it will now bedescribed in more detail, though by way of illustration only, withreference to the following examples:

GROUP I

Preparations of Derivatives of General Formula I via the MixedAnhydrides of General Formula II

Example 1

Preparation of N-acetyl-para-aminophenyl N'-acetyl-D,L-methionate (A)via the mixed anhydride.

Stage A: Preparation of the Mixed Anhydride (II, where n=2 and X'=Me)

To a mixture of 19.1 g of N-acetylmethionine and 9.45 g of methylchloroformate in 200 ml of chloroform 21 g of triethylamine were addeddropwise, while maintaining the temperature below 15° C. When additionwas complete, the mixture was refluxed for 15 minutes then allowed tocool to room temperature.

Stage B: Conversion of the Mixed Anhydride into the Methionate (A)

15.1 g of N-acetyl-para-aminophenol were added to the reaction productfrom Stage A and the mixture was refluxed for 1.5 hours. The mixture wasallowed to cool to room temperature, 100 ml of water are added and themixture was shaken vigorously until precipitation of the productoccurred.

The product was collected and dried in vacuum, to yield 13 g ofN-acetyl-para-aminophenyl N'-acetyl-D,L-methionate, m. pt.=183° C.

Example 2

Preparation of N-acetyl-para-aminophenyl N'-acetyl-S-methylcysteinate(E) via the mixed anhydride

Stage A: Preparation of the Mixed Anhydride (II, where n=1 and X'=Me)

To a mixture of 17.7 g of N-acetyl-S-methylcysteine and 9.45 g of methylchloroformate in 200 ml of chloroform 21 g of triethylamine was addeddropwise, while maintaining the temperature below 15° C. When additionwas complete, the mixture was refluxed for 15 minutes, then allowed tocool to room temperature.

Stage B: Conversion of the Mixed Anhydride into the Cysteinate (E)

15.1 g of N-acetyl-para-aminophenol were added to the reaction productobtained from Stage A, and the mixture was refluxed for 1.5 hours. Themixture was then allowed to cool to room temperature, and 100 ml. ofwater were added, and the mixture shaken vigorously until precipitationof the product occurred.

The product was collected and dried in vacuum, to yieldN-acetyl-para-aminophenyl N'-acetyl-S-methylcysteinate.

Example 3

Preparation of di-(N-acetyl-para-aminophenyl) N',N"-diacetylcystinate(C) via the mixed anhydride

Stage A: Preparation of the Mixed Anhydride (II, where n=1, X'=II', p=1and n'=1)

21.7 g (0.2 mole) of ethyl chloroformate in 100 ml of chloroform wereadded to a mixture of 32.4 g (0.1 mole) of N,N'-diacetylcystine and 20.2g (0.2 mole) of triethylamine in 250 ml of chloroform, over a period ofhalf to one hour, keeping the temperature at 20° C. Immediately afterthe addition of the ethyl chloroformate was completed, a further 4.04 g(0.04 mole) of triethylamine were added.

Stage B: Conversion of the Mixed Anhydride into the Cystinate (C)

The temperature of the reaction product obtained from Stage A wasadjusted to 20°-25° C., and 30.2 g (0.2 mole) ofN-acetyl-para-aminophenol were added portionwise. This mixture wasstirred at 25° C. for three hours. The product recovered by filtrationwas the desired di-(N-acetyl-para-aminophenyl) N',N"-diacetylcystinate.

Example 4

Preparation of N-acetyl-para-aminophenyl N'-acetylcysteinate (B) via themixed anhydride, with preliminary blocking and subsequent deblocking ofthe S--H bond

Preliminary Stage X: Complete Blocking of Starting Material

A mixture of 32.6 g of N-acetyl-L-cysteine, 18.0 g of dihydropyran and atrace of conc. hydrochloric acid was refluxed in 150 ml. ofdichloromethane for 40 minutes.

The solution was filtered and concentrated under reduced pressure, toyield 50 g of the desired tetrahydropyran derivative as a colorless oil.

Preliminary Stage Y: Partial Deblocking of Starting Material

A solution of 40 g of the tetrahydropyran derivative, obtained fromPreliminary Stage X above, dissolved in 55 ml of methanol, was addedwhile stirring to 250 ml. of 1M aqueous sodium hydroxide solution, andthe stirred mixture was kept at room temperature for 25 minutes.

The mixture was then extracted with ethyl acetate (to remove unreactedtetrahydropyran derivative) and the aqueous layer was neutralised with asolution of 15.2 g of acetic acid in 50 ml. of water. The aqueoussolution was re-extracted with ethyl acetate (2×100 ml), and the ethylacetate extracts were combined and dried over magnesium sulfate. Theethyl acetate solution was concentrated under reduced pressure, andafter stripping off residual solvents under high vacuum 7.8 g of thedesired S-tetrahydropyranyl-N-acetylcysteine were obtained as an oil.

Stage A: Preparation of the blocked Mixed Anhydride (II, where n=1, m=1,Q=C, Ayl=Et and X'=tetrahydropyranyl)

A stirred mixture of 7.2 g of theS-tetrahydropyranyl-N-acetyl-L-cysteine obtained above and 3.2 g ofethyl chloroformate in 75 ml of toluene was cooled to 10° C. and 3.2 gof triethylamine were slowly added at such a rate as to keep thetemperature in the range of 10°-15° C.

Stage B: Conversion of the Mixed Anhydride into the blocked Cysteinate(III, where n=1)

The reaction mixture was allowed to warm up to room temperature, andthen a solution of 4.4 g of N-acetyl-para-aminophenol and 1.45 g ofsodium hydroxide in 30 ml of water was added. The mixture was stirredvigorously for 15 minutes, then the aqueous and toluene layers weredecanted from the residual oil. The oil was dissolved in 50 ml of ethylacetate, washed with 10% aqueous sodium carbonate solution (2×100 ml),and dried over magnesium sulfate. Concentration of the ethyl acetatesolution under reduced pressure gave an oil, which crystallised fromchloroform-ether (1:2) at 0° C. overnight, yielding 1.5 g of the desiredN-acetyl-para-aminophenyl N'-acetyl-S-tetrahydropyranyl-L-cysteinate, m.pt.=170°-172° C.

Subsequent Stage Z: Deblocking to form the Cysteinate (B)

A solution of 0.5 g of N-acetyl-para-aminophenylN'-acetyl-S-tetrahydropyran-L-cysteinate in 80 ml of chloroform wasstirred vigorously with 1M hydrochloric acid for 6 hours. The chloroformlayer was then removed, dried over magnesium sulfate and concentratedunder reduced pressure to an oil. Trituration of the oil with etheryielded 0.32 g of the desired N-acetyl-para-aminophenylN'-acetyl-L-cysteinate, as a white solid, m. pt.=135°-138° C.

GROUP II

Preparation of Derivatives of General Formula I via thepara-nitrophenyl-substituted Intermediates of General Formula VIII

Example 5

Preparation of N-acetyl-para-aminophenyl N'-acetyl-D,L-methionate

Stage A: Preparation of para-nitrophenyl N-acetyl-D,L-methionate

A solution of 16.8 g of N-acetyl-D,L-methionate in 50 ml of pyridine wasadded to a solution of 28.5 g of bis(4-nitrophenyl) sulfite in 90 ml. ofpyridine; and the dark mixture was kept at room temperature for 1 hour.Concentration of the dark mixture under reduced pressure at atemperature below 40° C. gave a dark oil, which was dissolved in 100 mlof ethyl acetate and washed first with 2M hydrochloric acid then withsodium carbonate solution and finally with water, and then dried overmagnesium sulfate.

After removal of the ethyl acetate under reduced pressure 100 ml ofether were added to the residual oil. This afforded cream needles, whichwere removed and washed with ether and suction dried. Yield 17.2 g.

Stage B: Preparation of para-aminophenyl N-acetyl-D,L-methionate

A solution of 5 g of 4-nitrophenyl N-acetyl-D,L-methionate in 80 ml ofacetic acid was added slowly to a stirred mixture of 40 g of iron powderand 180 ml of hot acetic acid heated on a steam bath. After 1 hour themixture was filtered, and the acetic acid solution was concentratedunder reduced pressure to leave said product a brown gum.

Stage C: Preparation of N-acetyl-para-aminophenylN'-acetyl-D,L-methionate

The gum was treated with 20 ml of acetic anhydride and kept for 15minutes. 100 ml of water were added and the mixture was extracted twicewith ethyl acetate. The ethyl acetate solution was concentrated underreduced pressure and the residue was treated with 50 ml of ether andallowed to stand. This gave a coating of white solid around the flaskand an oil at the bottom of the flask.

The solid (2 g) was removed and recrystallised twice from ethylacetate/methanol, m. pt.=181° C.

It is contemplated that di-(N-acetyl-para-aminophenyl)N'-acetylcystinate (C), the di-(N-acetyl-para-aminophenyl)N',N"-diacetyl-cystathionate (D) or the N-acetyl-para-aminophenolN'-acetyl-S-methyl-cysteinate (E) each will be prepared in a mannersimilar to that described in Example 5 above, using molar equivalentquantities of the appropriate reactants in each instance.

In Examples 1, 2, 3 and 5 above both the starting material and the finalproduct were racemic mixtures of the D and L isomers; but it iscontemplated that by using the optically-active L-isomer as startingmaterial in each instance, the corresponding L-isomer of the finalproduct will be obtained.

So as to show another aspect of this invention details of theformulation of N-acetyl-para-aminophenyl N'-acetyl-D,L-methionate intopharmaceutical compositions will now be given, though again only forpurpose of illustration, as follows:

FORMULATION 1

Composition in the form of Tablets containing bothN-acetyl-para-aminophenol and N-acetyl-para-aminophenylN'-acetyl-D,L-methionate

A tabletting mixture was made up by intimately mixing together thefollowing ingredients:

    ______________________________________                                        N--Acetyl-para-aminophenol 600.00 g                                           N--Acetyl-para-aminophenyl N--acetyl-D,L-                                                                800.00 g                                           methionate                                                                    Maize starch               131.48 g                                           Potassium sorbate          1.16 g                                             Talc                       27.80 g                                            Stearic acid               5.56 g                                                                        1566.00 g                                          ______________________________________                                    

The mixture was then compressed by conventional tabletting machinery toform 780 mg tablets, each containing 300 mg of N-acetyl-para-aminophenoland 400 mg of N-acetyl-para-aminophenyl N'-acetyl-D,L-methionate.

FORMULATION 2

Composition in the form of Tablets containing bothN-acetyl-para-aminophenol and N-acetyl-para-aminophenyl N'-acetyl-D,L-methionate in combination with other active compounds.

A tabletting mixture was made up by intimately mixing together thefollowing ingredients:

    ______________________________________                                        N--Acetyl-para-aminophenol 540.00 g                                           N--Acetyl-para-aminophenyl N'--acetyl-D,L-                                                               720.00 g                                           methionate                                                                    2-(para-Chlorophenyl)-tetrahydro-3-methyl-                                                               200.00 g                                           4H--1,3-thiazin-4-one-1,1-dioxide                                             Gelatin                    12.60 g                                            Maize starch               64.00 g                                            Alginic acid               16.20 g                                            Talc                       42.00 g                                            Stearic acid               24.60 g                                            Magnesium stearate         4.60 g                                                                        1624.00 g                                          ______________________________________                                    

The mixture was then compressed by conventional tabletting machinery toform 812 mg tablets, each containing 270 mg ofN-acetyl-para-aminophenol, 360 mg of N-acetyl-para-aminophenylN'-acetyl-D,L-methionate, and 100 mg of2-(para-chlorophenyl)-tetrahydro-3-methyl-4H-1,3-thiazin-4-one-1,1-dioxide.

FORMULATION 3

Composition in the form of Tablets

A tabletting mixture was made up by intimately mixing together thefollowing ingredients:

    ______________________________________                                        First granulation                                                             N--Acetyl-para-aminophenol                                                                              240.00   g                                          N--Acetyl-para-aminophenyl N'--acetyl-D,L-                                                              320.00   g                                          methionate                                                                    Codeine phosphate         6.40     g                                          Sodium dioctyl sulfosuccinate 60% w/w solution                                                          1.312    g                                          Sorbitol                  8.40     g                                          Maize starch              32.00    g                                          Polyethylene glycols (av. mol. wt. 6000-7500)                                                           9.60     g                                          Second granulation                                                            Weight of granules from first granulation                                                               612.702  g                                          Talc                      36.71    g                                          Magnesium stearate        4.05     g                                          Maize starch              4.32     g                                          Sorbitol                  96.44    g                                          Finely divided silicon dioxide                                                                          2.89     g                                          (200 m.sup.2 /g - BET)                                                        ______________________________________                                    

The mixture was then compressed by conventional tabletting machinery toform 757 mg tablets, each containing 300 mg ofN-acetyl-para-aminophenol, 400 mg of N-acetyl-para-aminophenylN'-acetyl-D,L-methionate, and 8 mg. of codeine phosphate.

FORMULATION 4

Composition in the form of Soluble Tablets

A tabletting mixture was made up by intimately mixing together thefollowing ingredients:

    ______________________________________                                        N--Acetyl-para-aminophenol                                                                              300.00 g                                            N--Acetyl-para-aminophenyl N'--acetyl-                                                                  400.00 g                                            D,L-methionate                                                                Codein phosphate          8.00 g                                              Caffeine                  30.00 g                                             Sorbitol                  50.00 g                                             Sodium saccharin          10.00 g                                             Sodium bicarbonate        1500.00 g                                           Glutamic acid             0.90 g                                              Sodium lauryl sulfate     0.10 g                                              Anhydrous citric acid     925.00 g                                            Sodium carbonate          30.00 g                                             Magnesium stearate        0.60 g                                              ______________________________________                                    

The mixture was then compressed by conventional tabletting machinery toform soluble 3,254 mg tablets each containing 300 mg ofN-acetyl-para-aminophenol, 400 mg of N-acetyl-para-aminophenylN'-acetyl-D,L-methionate, 8 mg of codeine phosphate and 30 mg ofcaffeine.

FORMULATION 5

Composition in the form of Liquid Draught

The following ingredients were mixed together:

    ______________________________________                                        N--Acetyl-para-aminophenol                                                                             64.8    kg                                           N--Acetyl-para-aminophenyl N'--acetyl-                                                                 106.4   kg                                           D,L-methionate                                                                Ethyl alcohol            383.0   kg                                           Propylene glycol         466.0   kg                                           Glycerol                 1700.0  kg                                           Passion fruit flavour    45.0    kg                                           Eurocol Tartrazine       0.675   kg                                           Purified water q.s. to   4500.00 liters                                       ______________________________________                                    

to give a mixture containing 72 mg. of N-acetyl-para-aminophenol and 118mg of N-acetyl-para-aminophenyl N'-acetyl-D,L-methionate per 5 ml. ofsolution.

In all of Formulations 1-5 above it is possible of course to vary theamount of N-acetyl-para-aminophenyl N'-acetyl-D,L-methionate employed,with corresponding adjustment in the amounts of other ingredients--andsome or even all of the N-acetyl-para-aminophenylN'-acetyl-D,L-methionate can moreover be replaced by otherN-acetyl-para-aminophenyl N'-acetylaminothioalkanoate of general formulaI, specifically the preferred ones of structural formulae B, C, D, E andF hereinbefore identified, again if desired with appropriate adjustmentof the amounts of those compounds and the other ingredients.

Finally, as illustrative, certain evaluations will be given below whichindicate the freedom from undesirable side-effects and the excellentanalgesic properties of one of the compounds of this invention, namelyN-acetyl-para-aminophenyl N'-acetyl-D,L-methionate, of structuralformula A given hereinabove, which therefore will for convenience bereferred to below as "Compound A".

EVALUATION OF STABILITY

In order first to evaluate the stability of Compound A in thegastro-intestinal tract, simulated gastric and intestinal fluids andother test fluids were prepared as follows:

Preparation of Simulated Gastric Fluid

0.35 gram of sodium chloride, 0.5 gram of glycine and 94 ml of Mhydrochloric acid were mixed, and the volume was then made up to 1000 mlwith distilled water. The pH of this system was about 1.79.

Preparation of Simulated Intestinal Fluid

6.1 grams of monobasic potassium phosphate and 3.9 grams of disodiumhydrogen phosphate were dissolved in 1000 ml. of water. The pH of theresulting solution was 6.85.

Preparation of Tris Buffer

0.05M tris buffer was made by dissolving 6.05 grams oftris-(hydroxymethyl)-methylamine in 1000 ml. of water, and adjusting thepH to 7.2 by the addition of 0.1M HCl.

Preparation of Serum

The serum used in the assays was prepared by taking whole blood intoplain tubes, incubating for one hour at 37° C., and then centrifuging at2000 RPM for 10 minutes.

Methods of Evaluation

Standard solutions of 1×10⁻⁴ M Compound A in analytical grade methanol(0.01 ml) were prepared, and tested in the simulated gastric andintestinal fluids, in tris buffer alone, in tris buffer with added hogliver esterase and also in serum. The solutions under test wereincorporated in 1 ml. of each of the test fluids. A temperature of 30°C. was used for the assays involving the hog liver esterase, whereas atemperature of 37° C. was used for the assays involving the simulatedgastric and intestinal fluids and the serum. After this, the mixture wasallowed to stand and the supernatant liquid was spotted onto silicat.l.c. plates and developed in 90:10 chloroform/methanol. The mixturewas then extracted with 5 mls of chloroform and thechloroform-containing mixture was filtered through phase-separatingpapers, which allow only the chloroform solution to pass through. Thechloroform solution was also spotted onto silica t.l.c. plates anddeveloped in 90:10 chloroform/methanol

Results of Stability Evaluation

The results of this procedure, carried out with all the test fluidsmentioned above, show that:

1. at 30° C. the presence of hog liver esterase had no effect on therate of hydrolysis of Compound A. A half-life of about 475 minutes wasrecorded for Compound A in the tris buffer and also in the tris bufferplus the hog liver esterase;

2. at 37° C. the presence of human serum increased the rate ofhydrolysis of Compound A. Using serum from two volunteers, half-lives of140 and 150 minutes were recorded. The ability of human serum tohydrolyse N-acetyl-para-aminophenyl N'-acetyl-D,L-methionate indicatesthe presence of some specific catalytic factor which is absent from hogliver esterase. Hydrolysis of the ester in tris buffer at 37° C. gave ahalf-life of 240 minutes. This shows quite an increase in the rate ofspontaneous hydrolysis over a 7° C. rise in temperature; and

3. the hydrolysis of the ester in simulated gastric and intestinal juicewas very slow. Half-lives of 40 hours and 31.5 hours respectively wererecorded.

EVALUATION OF PHARMACOLOGICAL EFFECTS

In order to demonstrate and investigate the metabolism and resultantblood-levels of N-acetyl-para-aminophenol following administration ofCompound A in vivo, as compared with those obtained by administration ofcomparable amounts of N-acetyl-para-aminophenol itself, with or withoutadmixture of methionine, the following experiments were performed.

(A) Investigation of Serum Concentrations in Mice

36 Charles River COBS mice (18 males and 18 females) weighing 30 g eachwere deprived of food for 16 hours before the trial but were given theirusual drinking water. Three groups, each containing 6 males and 6females, received a dosage of 1 ml/100 g body weight per mouse, orallyby stomach tube, of a suspension in 1% gum tragacanth of respectively:

Group X: 500 mg/kg of N-acetyl-para-aminophenol.

Group Y: 500 mg/kg of N-acetyl-para-aminophenol+500 mg/kg of methionine.

Group Z: 1073 mg/kg of Compound A.

A blood sample was taken by throat-slitting from one male and one femalefrom each Group at each study time, i.e. 1/2, 1, 2, 4 and 24 hours afterthe oral administration.

At times 1/2, 1, 2 and 4 hours, each serum sample was divided into three0.1 ml trial samples for the following three determinations:

Determination 1:

This determination was carried out after acidic hydrolysis, according toan adaptation of the method of Heirwegh and Fevery [Clin. Chem., (1967),13, (3), 215-219] enabling a determination to be carried out on thesmall trial sample. This method records N-acetyl-para-aminophenol(abbreviated as "NAPA") in any form, thus as free NAPA, conjugated NAPAand NAPA chemically-bound in Compound A or any metabolites, and theresults set out below in Table I thus record "total NAPA" as measured bythis method.

                  TABLE I                                                         ______________________________________                                        Serum "total NAPA" (μg/ml) in                                              Charles River Mice                                                            Time in hours after oral                                                      administration     1/2 hr.                                                                              1 hr.  2 hrs.                                                                              4 hrs.                                 ______________________________________                                        500 mg/kg of NAPA                                                                            Male    217    263  250   107                                                 Female  336    262  147   374                                                 Mean    277    263  199   241                                  500 mg/kg of NAPA                                                                            Male    278    236  110   43                                   +              Female  402    128  70    36                                   500 mg/kg Methionine                                                                         Mean    340    182  90    40                                   1073 mg/kg Compound A                                                                        Male     96    255  67    94                                                  Female  110    131  70    44                                                  Mean    103    193  69    69                                   ______________________________________                                    

The results of Table I show that a significantly lower peak blood NAPAlevel (at 1/2 hour after administration) is achieved with Compound A,i.e., N-acetyl-para-aminophenyl N'-acetylmethionate or the ester ofN-acetyl-para-aminophenol (NAPA) and N-acetylmethionine, than thatobtained with either NAPA alone or NAPA in combination with methionine.

Determination 2:

This determination was carried out by direct extraction, with no priorhydrolysis, according to an adaptation of the above-mentioned method.This method thus records "free NAPA", and the results are set out inTable II below.

                  TABLE II                                                        ______________________________________                                        Serum "free NAPA" (μg/ml) in                                               Charles River Mice                                                            Time in hours after oral                                                      administration     1/2 hr.                                                                              1 hr.  2 hrs.                                                                              4 hrs.                                 ______________________________________                                        500 mg/kg of NAPA                                                                            Male    127    168  115   32                                                  Female  254    161  78    31                                                  Mean    191    165  97    32                                   500 mg/kg of NAPA                                                                            Male    215    102  44    MQC                                  +              Female  254    30   MQC   MQC                                  500 mg/kg of Methionine                                                                      Mean    235    66   ˜22                                  1073 mg/kg of  Male     67    78   MQC   MQC                                  Compound A     Female   66    74   MQC   MQC                                                 Mean     67    76                                              ______________________________________                                         [In Table II above, MQC = Value below the minimum quantifiable                concentration estimated at 20 μg/ml.]                                 

As shown in Table I, the results of Table II also show that asignificantly lower peak blood NAPA level (at 1/2 hr. afteradministration) is achieved with Compound A than that obtained witheither NAPA alone or NAPA in combination with methionine.

Determination 3:

This determination was carried out after 16 hours of incubation in thepresence of β-glucuronidase and arylsulfatase of a Helix Pomatia juice,and direct extraction of the liberated NAPA. Colorimetry was thencarried out according to the method quoted above. This method thusrecords what is here designated "enzymatic NAPA", including free NAPAand NAPA mainly conjugated to the glucuronides, and the results are setout in Table III below.

                  TABLE III                                                       ______________________________________                                        Serum "enzyme NAPA" (μg/ml) in                                             Charles River Mice                                                            Time in hours after oral                                                      administration     1/2 hr.                                                                              1 hr.  2 hrs.                                                                              4 hrs.                                 ______________________________________                                        500 mg/kg of NAPA                                                                            Male    221    254  241   106                                                 Female  392    254  146   368                                                 Mean    307    254  196   237                                  500 mg/kg of NAPA                                                                            Male    294    192  97    50                                   +              Female  392    135  64    42                                   500 mg/kg of Methion-                                                                        Mean    343    164  81    46                                   ine                                                                           1073 mg/kg of Compound                                                                       Male    101    246  67    98                                   A              Female  115    115  83    50                                                  Mean    108    181  75    74                                   ______________________________________                                    

Results

The results are given in Tables I to III above and can be interpreted asfollows:

NAPA administered on its own gives a high "total" or "enzymatic" serumNAPA concentration from the first 1/2 hour onwards after oraladministration. This concentration slowly decreases but remains high upto 4 hours after oral administration.

NAPA administered in admixture with methionine gives the same high serum"total" on "enzymatic" NAPA from the first half-hour onwards, but thisconcentration decreases more rapidly than when NAPA is administered onits own, showing that methionine accelerates the elimination of NAPA.Compound A does not however give the acute peak observed at one-half anhour after the administration of NAPA+methionine, although theconcentration of serum "total" or "enzymatic" NAPA equals that providedby the NAPA+methionine admixture one hour after oral administration, andthereafter the concentration of serum "total" or "enzymatic" NAPA thendecreases according to the same kinetics as those observed with theNAPA+methionine mixture. The results of the respective "total NAPA" and"enzymatic NAPA" determinations are virtually the same for each of thesamples tested, which means that NAPA is found in mouse serum, for eachof the substances tested, mainly in the form of free NAPA and NAPAconjugated to the glucuronides. From all this it can be concluded thatCompound A gives serum NAPA concentrations in mice identical to thoseobtained with an equimolecular mixture of NAPA and methionine, except atone half an hour after the oral administration of the substances, whenthe acute peak observed is significantly lower with Compound A than thatobserved with the NAPA+methionine mixture, suggesting significantly lessdanger of overdose liver damage at this stage with Compound A than withthe NAPA-methionine mixture.

(B) Investigation of Serum Concentrations in Beagle Dogs

6 Beagle dogs (3 males and 3 females) were dosed once a day (at 08.00hrs. each day) for 23 days with Compound A, suspended in 1% gumtragacanth, which was administered orally by oesophageal tube, at a rateof either 432 mg/kg (2 dogs, 1 male and 1 female) or 675 mg/kg (4 dogs,2 male and 2 female). During this period the dogs received their usualfood and water until 16.00 hrs. on the day before the test; but fromthat time until 08.00 hrs. on the day of the test they were given nofood, although they received their usual drinking water. The usualdosage of Compound A was however given to the animal at 08.00 hrs. onthe day of the test.

Without any further feeding, serum samples were then taken from eachanimal 2, 4 and 6 hours after the oral administration of Compound A,thus at 10.00 hrs., 12.00 hrs. and 14.00 hrs. on the day of the test.After the 6-hour serum sample had been taken, the animals received theirusual food for a while; but they were again made to fast for 12 hoursfrom 20.00 hrs. on the day of the test until 08.00 hrs. on the followingday, when the last, 24-hour serum sample was taken.

Each sample was divided, and the NAPA in each sample-portion wasdetermined in accordance with an adaptation of the method of Heirweghand Fevery [Clin. Chem., (1967), 13, (3), 215-219] which enablesdetermination to be carried out on study samples of 0.1 to 0.5 ml serum.

Determination 4:

A direct determination similar to that used in Determination 2 abovegave results for "free NAPA" as set out in Table IV below:

                  TABLE IV                                                        ______________________________________                                        Serum "total NAPA" (μg/ml) in beagle                                       dogs treated for 23 days with Compound A                                      Time in hours after                                                           oral administration                                                           of the last dose                                                              of Compound A    2 hrs.  4 hrs.  6 hrs.                                                                              24 hrs.                                ______________________________________                                        432 mg/kg of Male    104      84.8  73.2 11.8                                 Compound A   Female  104     115   110   30.2                                              Mean    104     100    92   21.0                                 675 mg/kg of Male     46.4   135   150   30.2                                 Compound A   Male    113     170   179   34.8                                              Female   76.8   135   106   22.8                                              Female  113     159   150   45.0                                              Mean     87     150   146   33.2                                 ______________________________________                                    

Determination 5:

A determination similar to that in Determination 1 above, carried outafter acid hydrolysis, gave results for "total NAPA", comprising freeNAPA, conjugated NAPA and NAPA related chemically to Compound A or anymetabolites, which are set out in Table V below

                  TABLE V                                                         ______________________________________                                        Serum "free NAPA" (μg/ml) in beagle                                        dogs treated for 23 days with Compound A                                      Time in hours after                                                           oral administration                                                           of the last dose of                                                           Compound A       2 hrs.  4 hrs.  6 hrs.                                                                              24 hrs.                                ______________________________________                                        432 mg/kg of Male    30.6    13.8   7.8  MQC                                  Compound A   Female  37.6    26.0  11.0  MQC                                               Mean    34.1    19.9   9.4                                       675 mg/kg of Male    11.4    14.6  12.6  MQC                                  Compound A   Male    37.6    36.2  15.3  MQC                                               Female  24.6    12.8   8.2  MQC                                               Female  37.6    22.6   8.2  MQC                                               Mean    27.8    21.6  11.1                                       ______________________________________                                         [In Table V above, MQC = value less than the minimum quantifiable             concentration which is estimated to be 4 μg/ml]                       

The results in Table V above demonstrate that Compound A is easilyhydrolyzed in this method.

From the results set out in Tables IV and V above, it will be seen thatNAPA is recovered in the serum in easily determinable amounts up to 6hours after the last oral administration of Compound A, in the two forms"free NAPA" and "total NAPA", and up to 24 hours in the single form"total NAPA".

(C) Investigation of Analgesic Activity in Rats

In accordance with the conventional Randal-Selitto technique, groups of12 female Charles River rats, 160-170 g in bodyweight, were injectedsubcutaneously in one hind-paw with yeast, and 21/2 hours later, whilethe inflammation was ongoing, either N-acetyl-para-amino-phenol [NAPA]or N-acetyl-para-aminophenyl N'-acetyl-D,L-methionate [Compound A] wasthen administered orally, at a rate of either 1 or 2 millimoles/kg. Acontrol group received only the gum tragacanth vehicle.

In order to evaluate the effect of the treatment upon pain-thresholds,pressure was applied both to the injected hind-paw and to the othernon-injected hind-paw. The pain-threshold was noted as the pressure atwhich the rat flinched, measured using an Ugo Basile "Analgesimeter" forboth hind-paws of each rat at intervals of 1 hour, 3 hours, 5 hours and22 hours after the administration, and expressed as the percentage ofthe pain-threshold pressure for the injected (and thus inflamed)hind-paw as against the non-injected (and thus healthy) hind-paw.

The results (after statistical treatment by the t-test versus thecontrol groups) are summarized in Table VI below:

                                      TABLE VI                                    __________________________________________________________________________    Percentage Pain-Thresholds in the                                             Randall-Solitto Test in Rats                                                                     Time from Administration                                                      of Compound Tested                                                            (Time from Injection of Yeast)                             Compound                                                                             Amount in                                                                           Amount in                                                                           1 hr.                                                                              3 hrs.                                                                             5 hrs.                                                                             22 hrs.                                     Administration                                                                       Millimoles                                                                          mg/kg (3.5 hrs.)                                                                         (5.5 hrs.)                                                                         (7.5 hrs.)                                                                         (24.5 hrs.)                                 __________________________________________________________________________    Control                                                                              --    --    41 ± 3.1                                                                        42 ± 2.9                                                                        44 ± 4.1                                                                        42 ± 3.5                                 NAPA   1     151   45 ± 3.5                                                                        48 ± 4.1                                                                        47 ± 1.7                                                                        43 ± 3.4                                 `A`    1     324   44 ± 3.8                                                                        41 ± 2.9                                                                        50 ± 4.2                                                                        50 ± 2.4                                 NAPA   2     302   50 ± 4.2                                                                        54 ± 4.0                                                                        53 ± 3.9                                                                        54 ± 4.7                                                         P<0.05                                                `A`    2     648   53 ± 3.3                                                                        54 ± 3.2                                                                        59 ± 2.7                                                                        54 ± 1.9                                                    P<0.02                                                                             P<0.02                                                                             P<0.01                                                                             P<0.01                                      __________________________________________________________________________

The results shown in Table VI above indicate thatN-acetyl-para-aminophenyl N'-acetyl-D,L-methionate (Compound A) has astatistically-significant analgesic effect at the 2 millimoles/kg level,which is at least as great as N-acetyl-para-aminophenol (NAPA) judgingfrom the level of the statistical significance reached.

(D) Investigation of Antipyretic Activity in Rats

Using the conventional procedure for the yeast-induced hyperthermiatest, several groups of 10 male Charles River rats were given asubcutaneous injection of dried yeast. Immediately after the injectionthey were then medicated orally with respectively:

100 mg/kg of N-acetyl-para-aminophenol (NAPA);

90 mg/kg of NAPA+21.5 mg/kg of Compound A;

80 mg/kg of NAPA+43.0 mg/kg of Compound A; and

60 mg/kg of NAPA+86.0 mg/kg of Compound A.

In the mixtures the amount of Compound A, N-acetyl-para-aminophenylN'-acetyl-D,L-methionate, used was in each case equimolar with the 10%,20% or 40% reduction in the amount of NAPA, so that all the medicationswere carried out at a level equivalent to 100 mg/kg of NAPA.

Results

The body temperature of each rat was carefully monitored for 6 hoursfollowing oral administration of the NAPA and NAPA/Compound A mixtures,and the results obtained showed that the four treatments had similarantipyretic effects, each starting at about 39.7° C. and all ranging atabout 37.8° to 38.0° C. after 1 hour, about 38.2°-38.6° C. after 2hours, about 38.9°-39.5° C. after 3 hours and about 39.5°-39.6° C. after4 hours. It can therefore be concluded that the antipyretic effect ofCompound A, N-acetyl-para-aminophenyl N'-acetyl-D,L-methionate, isvirtually identical upon an equimolar basis of comparison with that ofthe well-dried analgesic NAPA, N-acetyl-para-aminophenol.

(E) Investigation of Acute Oral Toxicity in Mice

Charles River (CD-1 COBS) albino mice, 20-22 g in weight, were kept forsome days in an air-conditioned animal house and fed with a standarddiet (sterile VAR granules). They were fasted overnight beforeexperiment, with water ad libitum. 15 male mice and 15 female mice wereused for each experiment.

N-Acetyl-para-aminophenol (NAPA) and N-acetyl-para-aminophenylN'-acetyl-D,L-methionate (Compound A) were then administered to themice, orally, as a suspension (shaken continuously with anelectromagnetic agitator) in 5% gum arabic at a volume of 0.02 ml/g,either individually or in variously proportioned mixtures and atdifferent dose levels, as shown in Table VII below.

                  TABLE VII                                                       ______________________________________                                        NAPA/Com-  N--acetyl-para-   N--acetyl-para-                                  pound A    aminophenol       aminophenyl N'--                                 Ratio      [NAPA]            acetyl-D,L-methionate                            (where applicable)                                                                       in mg/kg    +     [Compound A] in mg/kg                            ______________________________________                                        not applicable                                                                           350         +     0                                                [100% NAPA]                                                                              700         +     0                                                           1400        +     0                                                450/100    350         +     77.8                                                        700         +     155.6                                                       1400        +     311.1                                            400/200    400         +     200                                                         800         +     400                                                         1600        +     800                                              300/400    450         +     600                                                         900         +     1200                                                        1800        +     2400                                             not applicable                                                                           0           +     2000                                             [100% Compound A]                                                                        0           +     4000                                             ______________________________________                                    

A control group received only the 5% gum arabic vehicle. The mice wereobserved for 7 days, their behavioural patterns and mortality beingrecorded.

Results

The behaviour of the mice treated at the highest dose level of thevarious mixtures was similar to that observed in the mice medicated withN-acetyl-para-aminophenyl N'-acetyl-D,L-methionate alone at the 4 g/kgdose level and with N-acetyl-para-aminophenol alone at the 1400 mg/kgdose level. The animals developed hypothermia, tremor, tail extension,epistaxis and acrocyanosis, followed by death.

The animals which died at lower dose levels did not however presentthese symptoms.

The LD₅₀ 's at 24 hours and 7 days were calculated by the Miller andTainter method, expressing the LD₅₀ for the mixture as the totalquantity of N-acetyl-para-aminophenol given to the animal, assuming thatall the N-acetyl-para-aminophenol from Compound A is available, and thuson the basis that 100 mg of N-acetyl-para-aminophenylN'-acetyl-D,L-methionate gives 46.6 mg of N-acetyl-para-aminophenol. Theresults thus calculated are set out in Table VIII below.

                  TABLE VIII                                                      ______________________________________                                        NAPA/Com-                                                                              LD.sub.50 expressed in N--                                                                    Ratio of "total NAPA"                                pound A  acetyl-para-aminophenol                                                                       [derived both from                                   Ratio    (NAPA) mg/kg    NAPA proper and from                                 (where appli-                                                                          after     after     Compound A] relative                             cable)   24 hours  7 days    to Methionine                                    ______________________________________                                        not applicable                                                                         730 ± 65                                                                             690 ± 65                                                                             ∞                                          [100%                                                                         NAPA]                                                                         450/100  1000 ± 65                                                                            800 ± 70                                                                             10:1                                             400/200  1040 ± 135                                                                           930 ± 90                                                                             5:1                                              300/400  1730 ± 185                                                                           1500 ± 115                                                                           5:2                                              not applicable                                                                         ≧4000                                                                            ≧4000                                                                            0                                                [100% Com-                                                                    pound A]                                                                      ______________________________________                                    

From the results set out in Table VIII above it is readily apparent notonly that the LD₅₀ of Compound A, N-acetyl-para-aminophenylN'-acetyl-D,L-methionate, is several times greater than that of thewell-tried analgesic NAPA, N-acetyl-para-aminophenol, but furthermorethat the presence of even a quite minor proportion of Compound A inmixtures thereof with NAPA brings about a substantial increase in theLD₅₀ of such mixtures as compared with NAPA, thus demonstrating thehepatic safety of such compositions.

(F) Investigation of Acute Oral Toxicity in Rats

The investigation was carried out upon groups of 10 male and 10 femalealbino rats (Charles River CD-1 COBS) of 80-100 g body weight. The ratswere kept for some days in an air-conditioned animal house, and fed witha standard diet and drinking water ad libitum. Before the experimentthey were fasted overnight, with water ad libitum. NAPA and Compound Awere then administered orally by oesophageal intubation as suspensions(continuously shaken with an electromagnetic agitator) in 1% gum arabicat a volume of 2 ml/100 g body weight, and at dose levels of 1, 2, 4 and8 g/kg. A control group received orally, under the same experimentalconditions, merely 2 ml/100 g of the suspension vehicle. The rats werethen kept under observation during seven days, while their behaviourpatterns were observed and the mortality recorded

As regards behaviour, no alteration was observed in the behaviouralpatterns of the rats treated with Compound A--but administration of NAPAinduced complete prostration and hypothermia in all the rats at levelsof 2, 4 and 8 g/kg, half an hour after the administration. Hair erectionand continual watering of the eyes appeared two hours after theadministration. The first deaths were noticed eight hours after theadministration at 8 g/kg dose level, with convulsions of tetanic type,epistaxis, blood tears and acrocyanosis.

As regards mortality, in the case of Compound A no mortality wasrecorded at any of the different dose levels, as appears from Table IXbelow. Heavy mortality was however recorded in the case of NAPA,especially at the higher dose levels as appears from Table X below.

                  TABLE IX                                                        ______________________________________                                        Acute Toxicity Percentage Mortality                                                 Num-                                                                    Com-  ber                                                                     pound and     24      48    3    4    5    6    7                             A     Sex     Hours   Hours Days Days Days Days Days                          ______________________________________                                        8 g/kg                                                                              10 M    0       0     0    0    0    0    0                                   10 F    0       0     0    0    0    0    0                                   M & F   0       0     0    0    0    0    0                             4 g/kg                                                                              10 M    0       0     0    0    0    0    0                                   10 F    0       0     0    0    0    0    0                                   M & F   0       0     0    0    0    0    0                             2 g/kg                                                                              10 M    0       0     0    0    0    0    0                                   10 F    0       0     0    0    0    0    0                                   M & F   0       0     0    0    0    0    0                             1 g/kg                                                                              10 M    0       0     0    0    0    0    0                                   10 F    0       0     0    0    0    0    0                                   M & F   0       0     0    0    0    0    0                             CON-  10 M    0       0     0    0    0    0    0                             TROL  10 F    0       0     0    0    0    0    0                                   M & F   0       0     0    0    0    0    0                             ______________________________________                                    

                  TABLE X                                                         ______________________________________                                        Acute Toxicity Percentage Mortality                                                 Num-                                                                          ber                                                                           and     24      48    3    4    5    6    7                             NAPA  Sex     Hours   Hours Days Days Days Days Days                          ______________________________________                                        8 g/kg                                                                              10 M    90      100   100  100  100  100  100                                 10 F    100     100   100  100  100  100  100                                 M & F   95      100   100  100  100  100  100                           4 g/kg                                                                              10 M    90      90    90   90   90   90   90                                  10 F    70      70    70   70   70   70   70                                  M & F   80      80    80   80   80   80   80                            2 g/kg                                                                              10 M    10      10    10   10   10   10   10                                  10 F    0       0     0    0    0    0    0                                   M & F   5       5     5    5    5    5    5                             1 g/kg                                                                              10 M    10      10    10   10   10   10   10                                  10 F    0       0     0    0    0    0    0                                   M & F   5       5     5    5    5    5    5                             CON-  10 M    0       0     0    0    0    0    0                             TROL  10 F    0       0     0    0    0    0    0                                   M & F   0       0     0    0    0    0    0                             ______________________________________                                    

From the results in Tables IX and X above the LD₅₀ s at 24 hours and 7days for both Compound A and NAPA were calculated by the Miller andTainter method, on logarithmic paper, and were as follows:

Compound A: LD₅₀ (24 hrs. and 7 days)>8 g/kg.

NAPA: LD₅₀ (24 hrs.)=3.1±0.5 g/kg; LD₅₀ (7 days)=2.85±0.4 g/kg.

From this it can be concluded that Compound A when administered orallyin 5% gum arabic to the albino COBS rats proved far less toxic thanNAPA, and did not induce any mortality at dose levels up to 8 g/kg.

We claim:
 1. The mixed anhydride having the general formula ##STR23##wherein n is 1 or 2, m is zero or 1, Q represents either a carbon atomor a sulfinyl group S═O where m is zero or a carbon atom when m is 1,"Ayl" represents an alkyl group with 1-20 carbon atoms, an aryl groupwith 6-20 carbon atoms, an alkaryl group with 7-20 carbon atoms or anaralkyl group with 7-20 carbon atoms, and X' is a hydrogen atom,tetrahydro-2-pyranyl, a methyl group, or a mixed anhydride groupconforming to the general formula ##STR24## wherein Q and Ayl both havetheir previously-indicated meanings, n' is 1 or 2, m' is zero or 1, andp is zero or
 1. 2. The mixed anhydride having the formula ##STR25##wherein n is 1 or 2, "Ayl" represents an alkyl group with 1-20 carbonatoms, an aryl group with 6-20 carbon atoms, an alkaryl group with 7-20carbon atoms or an aralkyl group with 7-20 carbon atoms and X' is ahydrogen atom or a methyl group.